The present invention relates to a miniature, differential-type sensor being able to quickly measure levels of carbon dioxide dissolved in liquid solutions, which functions with a working electrode composed of unbuffered hydrogel and a pH-sensitive gas-permeable membrane, and a reference electrode composed of buffered hydrogel and a pH-sensitive gas-permeable membrane. More particularly, the present invention relates to the introduction of carbonic anhydrase into the unbuffered hydrogel to reduce the hydration time of carbon dioxide, thereby quickly measuring carbon dioxide levels, and the introduction of a controlled content of bicarbonate ions into the unbuffered hydrogel to improve the sensitivity of the sensor.
Quantification of carbon dioxide levels can be applied to various fields. For example, it can be applied for analyzing clinical samples in the medical diagnostic fields, for regulating combustion processes in chemical analysis fields, for diagnosing the severity of the green house effect, and for measuring the indexes related to aquatic ecosystem in an environmental field. The exact measurement of dissolved carbon dioxide becomes increasingly significant.
In a human body, carbon dioxide is present in a small quantity as a metabolic end product, resulting mainly from the metabolism of foods. When the partial pressure of carbon dioxide in blood is 40 mmHg or higher, carbon dioxide is released from erythrocytes. The free carbon dioxide is dissolved in blood plasma and hydrated to form carbonic acid (H2CO3), which is in turn dissociated into hydrogen ion (H+) and bicarbonate ion (HCO3+). Since the total content of carbon dioxide (CO2 gas, H2CO3, HCO3xe2x88x92, CO32xe2x88x92) in blood plasma affects the acid-base balance and pH of blood, as well as being an index for pulmonary ventilation, alveolar gas exchange capacity, and the quantity of the gas transferred to somatic tissues from blood, it is very important to accurately measure the levels of carbon dioxide dissolved in blood.
Carbon dioxide also plays an important role in the field of the ecosystem. For example, carbon dioxide is in an equilibrium state between air and water. In natural water, bicarbonate ions (HCO3xe2x88x92) act as a buffering reagent and keep the pH constant. As the concentration of carbon dioxide in the water is closely related to the health of aquatic ecosystems, it is important to accurately measure levels of carbon oxide dissolved in water in order to detect changes of the aquatic ecosystem.
For measuring concentrations of carbon dioxide, two types of carbon dioxide gas sensors are known: a Severinghaus-type carbon dioxide gas sensor, wherein an external reference electrode, a pH-sensitive electrode and a gas-permeable membrane are simultaneously housed in one sensor body; and a differential-type carbon dioxide gas sensor, wherein a working electrode and a reference electrode are separated in different sensor body.
As shown in FIG. 1, the Severinghaus-type carbon dioxide gas sensor comprises an external reference electrode 14, a working electrode 16 with pH-sensitive membrane 11, a gas-permeable membrane 12, and an unbuffered solution 13 in one sensor body 17. When the Severinghaus-type carbon dioxide gas sensor is immersed in a sample solution 18 of interest, a potential is generated by the sensor and displayed on a voltmeter 15.
Such a Severinghaus-type carbon dioxide gas sensor suffers from difficulty in the fabrication and miniaturization of the sensor, because the reference electrode is incorporated inside the sensor. In addition, another disadvantage of Severinghaus-type carbon dioxide gas sensor is that it cannot be used when the level of carbon dioxide is low. That is, at a low level of carbon dioxide, the Severinghaus-type carbon dioxide sensor is so slow in sensing rate and recovery rate that it cannot be used in automatic gas sensing systems. Furthermore, Severinghaus-type carbon dioxide gas sensor suffers from the disadvantage of being poor in detection limit.
FIG. 2 illustrats a differential-type carbon dioxide gas sensor, characterized in that a working electrode is separated from a reference electrode. The differential-type carbon dioxide gas sensor comprises a working electrode 20 composed of an unbuffered inner reference solution 13 and a pH-sensitive gas-permeable membrane 19; and a reference electrode 21 composed of a buffered inner reference solution 22 and the same pH-sensitive gas-permeable membrane 19 as that in a working electrode.
In the differential-type carbon dioxide gas sensor, charge separation and the accompanying potential difference occur at 4 different sites: Eouter1 between the pH-sensitive gas-permeable membrane 19 of the working electrode 20 and the sample solution 18; Eouter2 between the pH-sensitive gas-permeable membrane 19 of the reference electrode 21 and the sample solution 18; Einner1 between the pH-sensitive gas-permeable membrane 19 of the working electrode 20 and the unbuffered inner reference solution 13; and Einner2 between the pH-sensitive gas-permeable membrane 19 of the reference electrode 21 and the buffered inner reference solution 22. When such charge separations occur, Eouter1 and Eouter2 have the same value and thus can be counterbalanced, as the same pH-sensitive gas-permeable membranes are used. On the other hand, the charge separation Einner2 between the pH-sensitive gas-permeable membrane 19 of the reference electrode 21 and the buffered inner reference solution 22 is maintained at a constant value as the reference solution is buffered. Therefore, a change in carbon dioxide levels in a sample solution 18 causes only the charge separation Einner1 between the pH-sensitive gas-permeable membrane 19 and the unbuffered inner reference solution 13 of the working electrode 20, so that the resulting potential change enables the carbon dioxide levels of the sample solution to be quantitatively detected.
However, the conventional differential-type carbon dioxide gas sensor also suffers from long period of response time for sensing and recovery at a low level of carbon dioxide. Also, its detection limits are still not satisfactory. However, the differential-type carbon dioxide gas sensors are easier to miniaturize than the Severinghaus-type one.
Microchip-based carbon dioxide gas sensor may be fabricated in small sizes since all parts thereof including electrolyte layers can be fabricated as a layered structure. It can be also used as a multi-sensor capable of detecting various ions and gas species simultaneously with one chip. Additionally, the mass production of the microchip-based sensor can be achieved, resulting in reduced production cost. Furthermore, the small size of its sensing site makes it possible to detect even a trace amount of samples.
In this invention, we combined two advanced technologies in an attempt to obtain a planar microchip-based carbon dioxide sensing device with faster-preconditioning and response characteristics for being dissolved carbon dioxide measurement in physiological samples: one is a differential sensing arrangement to facilitate the micro-fabrication of potentiometric carbon dioxide electrodes, and the other is the use of carbonic anhydrase to shorten total measurement time. The pH-sensitive polymeric membranes adapted for use in constructing a differential carbon dioxide sensor system in this work function as both a gas-permeable membrane and an internal pH-sensing element. In the differential configuration, the carbon dioxide electrode is made with an unbuffered recipient layer including carbonic anhydrase, hence the pH changes are promoted and detected. The reference electrode, on the other hand, employs a strongly buffered hydrogel layer; therefore diffused carbon dioxide cannot change the pH in the recipient layer. In addition, the pH and ion response signals of the outer membrane surfaces (on the sample side) at both the carbon dioxide and the reference electrodes are identical, therefore they cancel out.
Therefore, it is an object of the present invention to provide a miniaturized differential-type carbon dioxide gas sensor comprising all parts of the sensor including electrolyte layers fabricated as a dried layered structure, such that it can be miniaturized.
Another object of the present invention is to provide a composition of unbuffered hydrogel membrane of the differential-type carbon dioxide gas sensor.
The above objects and other objects described in the detailed description of the present invention could be accomplished by the provision of a differential-type carbon dioxide gas sensor, comprising:
a) a working electrode composed of unbuffered hydrogel and pH-sensitive gas-permeable membrane, wherein carbonic anhydrase is incorporated into a unbuffered hydrogel; and,
b) a reference electrode composed of buffered hydrogel and pH-sensitive gas-permeable membrane.
The present invention also provides a composition of unbuffered hydrogel of a working electrode, which comprises: sodium bicarbonate; sodium chloride (or potassium chloride); and hygroscopic hydrogel and carbonic anhydrase.